• 한국가축번식학회지
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• 제20권4호
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• pp.467-472
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• 1997

본 연구는 체내생산 공핵배 핵이식의 최적 통전전압을 결정하기 위하여 그리고 공핵배의 질이 핵이식난자의 융합과 체외발달에 미치는 영향을 조사하기 위하여 수행되었다. 수핵난자는 체외성숙후 25∼27시간에 제핵하였고 난자의 추가성숙을 위해서 융합전에 18∼20시간 더 배양하였다. 상실배시기의 공핵배는 다배란 처녀우에서 채란한 후 질에 따라 양질과 저질로 구분하여 공시하였다. 공핵배의 핵이식은 체외성숙후 42∼44시간에 행하였고, 융합은 0.75kV/cm 또는 1.0kV/cm DC 전압으로 체외성숙 후 43∼45시간에 실시하였다. 융합율은 두 전안갑에 차이가 없었으나 난할율과 M＋B 발달율은 19.0%와 29.4%였다. 공핵배의 질은 융합율과 난할율에 크게 영향이 없었으나 저질공핵배의 핵이식으로 부터는 상실배의 발달이 없었다. 본 결과에서 체내생산 공핵배 핵이식의 최적전압은 1.0kV/cm DC이었으며 공핵배의 질은 핵이식배 발달에 영향하는 중요 요인 중에 하나이었다.

• 한국수정란이식학회지
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• 제24권3호
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• pp.231-235
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• 2009

Oxygen consumption has been regarded as a useful indicator for assessment of mammalian embryo quality. However, there was no standard criterion to measure the oxygen consumption of embryos. Here, we measured oxygen consumption of bovine embryos at various developmental stages was measured using a scanning electrochemical microscopy (SECM). We found that the oxygen consumption significantly increased in blastocyst-stage embryos compared to other stage embryos (from 2-cell-stage to morula-stage), indicating that oxygen consumption reflects the cell number ($5.2{\sim}7.6{\times}10^{14}/mol\;s^{-1}$ versus $1.2{\sim}2.4{\times}10^{14}/mol\;s^{-1}$, p<0.05). In the morula-stage embryos, the oxygen consumption of in vivo derived embryos was significantly higher than that of in vitro produced embryos ($4.0{\times}10^{14}/mol\;s^{-1}$ versus $2.4{\times}10^{14}/mol\;s^{-1}$, p<0.05). However, there was no significant difference in consumption of oxygen by in vivo and in vitro-derived bovine blastocyst-stage embryos (p>0.05). In the frozen-thawed blastocyst-stage embryos, live embryos showed significantly higher oxygen consumption than dead embryos ($4.7{\times}10^{14}/mol\;s^{-1}$ versus $1.0{\times}10^{14}/mol\;s^{-1}$, p<0.05). These results indicate that the measuring oxygen consumption by SECM can be used to evaluate bovine embryo quality.

• Journal of Animal Science and Technology
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• 제65권4호
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• pp.767-778
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• 2023

The aim of the research is to identify that porcine oocytes can function as recipients for interspecies cloning and have the ability to develop to blastocysts. Furthermore each mitochondrial DNA (mtDNA) in interspecises cloned embryos was analyzed. For the study, mouse-porcine and porcine-porcine cloned embryos were produced with mouse fetal fibroblasts (MFF) and porcine fetal fibroblasts (PFF), respectively, introduced as donor cells into enucleated porcine oocytes. The developmental rate and cell numbers of blastocysts between intraspecies porcine-porcine and interspecies mouse-porcine cloned embryos were compared and real-time polymerase chain reaction (PCR) was performed for the estimate of mouse and porcine mtDNA copy number in mouse-porcine cloned embryos at different stages.There was no significant difference in the developmental rate or total blastocyst number between mouse-porcine cloned embryos and porcine-porcine cloned embryos (11.1 ± 0.9%, 25 ± 3.5 vs. 10.1 ± 1.2%, 24 ± 6.3). In mouse-porcine reconstructed embryos, the copy numbers of mouse somatic cell-derived mtDNA decreased between the 1-cell and blastocyst stages, whereas the copy number of porcine oocyte-derived mtDNA significantly increased during this period, as assessed by real-time PCR analysis. In our real-time PCR analysis, we improved the standard curve construction-based method to analyze the level of mtDNA between mouse donor cells and porcine oocytes using the copy number of mouse beta-actin DNA as a standard. Our findings suggest that mouse-porcine cloned embryos have the ability to develop to blastocysts in vitro and exhibit mitochondrial heteroplasmy from the 1-cell to blastocyst stages and the mouse-derived mitochondria can be gradually replaced with those of the porcine oocyte in the early developmental stages of mouse-porcine cloned embryos.

• Plant Resources
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• 제7권2호
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• pp.87-92
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• 2004

Five different poplar hybrids were tested for rescuing embryo to elongate in vitro plantiets after hybridization. Ovaries and ovules were cultured on Woody Plant Medium (WPM) supplemented with cytokinins, 6-benzylamine (BA) and zeatin. Multiple shoots were initiated from half section of capsule with immature embryos after 21 days from pollination and tiny shoots were formed after the expansion of cotyledons in ovule cultures. Germinating response was better in intraspecific hybrids $(6.53\pm1.66)$ than interspecific crosses $(0.93\pm0.54)$ from half section of capsules on WPM medium. In general, zeatin was better than BA in inducing multiple shoots from isolated ovules. The highest average number $(19.40\pm4.53)$ of shoots was produced from immature ovules of 21 days post-pollination of WPM medium supplemented with 5.0 mg/L zeatin. The highest percentage of germination was 93% from the half section of in vitro ovary cultures. Soil acclimation was successfully conducted in cell tray containing artificially mixed soil with 96% survival rate.

• 한국수정란이식학회지
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• 제10권3호
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• pp.229-236
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• 1995

The experiments reported here take advantage of the large number of in vitro matured and in vitro fertilized(IVM /IVF) bovine oocytes which can be produced, permitting the design of controlled experiments to establish a simple defined medium for the study of early embryo requirements. A total of 1,386 IVM /IVF oocytes were used to compare a simple defined medium(KSOM) with more complex culture conditions used successfully for culture of bovine embryos but do not permit study of specific requirements. All experiments were extensively replicated factorials. In Experiment 1, KSOM was superior to Menezo B$_2$ medium in producing morulae plus blastocysts from IVM /IVF oocytes(33 vs 20%, P<0.()5). The yield of morulae plus blastocysts with KSOM was 22% and with BRLC added was 30%. In Experiment 2, (a 2x2 factorial of KSOM with or without BRLC and 0, 1 ng /ml of platelet derived growth factor, PDGF) more morulae plus blastocysts (40%) were produced in KSOM-BRLC co-culture containing 1 ng /ml PDGF than in the control KSOM(12%). In Experiment 3, there was no dose response when 0, 1 and 5 ng /ml of PDGF were added. The results with simple defined KSOM medium are sufficiently promising to indicate that specific requirements of the embryo may be examined in future studies with KSOM as a base.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2002년도 춘계학술발표대회 발표논문초록집
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• pp.39-39
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• 2002

The mechanisms underlying the visual assessment and resulting in optimum embryonic development following in vitro maturation, fertilization, and culture are unclear. It is known that in vitro produced embryos show more frequent occurrence of fragmentation, which result in poor developmental potential and decreased implantation rate. The objective of this study was to investigate the apoptotic rates in in vitro fertilization (IVF) and nuclear transferred (NT)bovine blastocyst. (omitted)

• 한국약용작물학회지
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• 제12권5호
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• pp.384-390
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• 2004

An in vtro nucellar polyembryo propagation method was established with mature seed of the Citrus junos Sieb. 7-8 nucellar polyembryos per seed were induced on MS basal medium without plant growth regulators. The polyembryos developed to complete plantlets on teatment with IBA. These shoots grew further in MS medium without plant growth regulators. Rooting of shoots occurred on MS medium supplemented with IBA. These plantlets were successfully transplanted to small plastic pot containing soil mixture. Somatic embryos were induced from nucellar polyembryo and maturation occurred spontaneously from proliferating cultures on MS medium without growth regulators. Random Amplified Polymorphic DNA (RAPD) marker analysis of in vitro and in vivo grown junos orange showed identical polymorphism indicative of their genetic stability. The RAPD polymorphism produced revealed same banding pattern in each regenerant. Hence, propagaton of junos orange by nucellalr polyembryos was efficient and produced in genetically stable plants under in vitro conditions.

• 한국수정란이식학회지
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• 제33권3호
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• pp.169-175
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• 2018

It has been claimed that artificial insemination (AI) of cows with frozen-thawed semen treated with commercially produced kits, Wholemom (in favour of female gender) increases the birth chance of calves with desired sex ratio by approximately 85% without decrease of pregnancy rates. Hence, this study was conducted to investigate the efficacy of wholemom kits as combined with frozen-thawed bovine semen during in vitro fertilization on the in vitro fertilization and developmental efficiency and sex ratios such as some reproductive parameters in bovine. For this, 1,737 oocytes were in vitro fertilized and developed. Agglutination effects on bovine after treatment of Wholemom kit were observed by time passage and dose respectively. To determine sex of embryos, Bovine embryo Y-specific gene primers(ConEY) and Bovine specific universal primer(ConBV) were used as multiple PCR method. Fertilization rate of wholemom-treated group was significantly lower than its of control group[66.9% (1,156/1,737) in Wholemom-treated group; 75.0% (610/813) in control group]. However, developmental rate after fertilization of both wholemom-treated and control groups were not significantly different [26.1% (404/1,156) in Wholemom-treated group; 27.4% (224/610) in control group]. Sex ratio of in vitro fertilized embryo with frozen-thawed semen treated with wholemom kit was determined by multi PCR. Female ratio in wholemom-treated group [85.4% (173/201)] was significantly higher than its of control group [47.2% (66/141)]. In conclusion, wholemom treatments of semen used in the in vitro fertilization and development of bovine oocytes provided increase in female ratio with decrease of fertilization rate.

• 한국수정란이식학회지
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• 제12권3호
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• pp.259-267
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• 1997

To improve the efficiency of production of cloned embryos and animals by nuclear transplantation in the rabbit, the effect of cell cycle of donor nuclei and type of recipient cytoplasm on the in vitro developmental potential and production efficiency of offspring was determined. The embryos of 16-cell stage were collected from the mated does at 48h post-hCG injection and they were synchronized to G$_1$ phase of 32-cell stage. The oocytes collected at 14h post-hCG injection were freed from cumulus cells and then enucleated. One group of the enucleated cytoplasms was activated by electrical stimulation prior to injection of donor nucleus, and the other group was not pre-activated. The separated G$_1$phase blastomeres of 32-cell stage embryos were injected into the perivitelline space of recipient cytoplasms. After culture for 20h post-hCG injection, the nuclear transplant oocytes were electrofused and activated by electrical stimulation and the fused nuclear transplant embryos were co-cultured for 120h and the nuclear transplant embryos developed to blastocyst stage were stained with Hoechst 33342 dye and their blastomeres were counted. Some of the nuclear transplant embryos developed in vitro to 2- to 4-cell stage were transferred into the oviducts of synchronized recipient does. The electrofusion rate was similar between the types of donor nuclei and recipient cytoplasms used. However, the nuclear transplant embryos using G$_1$ phase donor nuclei were developed to blastocyst at higher rate(60.3%) than those using S phase ones(24.7%). Also, when non-preactivated oocytes were used as recipient cytplasms, the develop-mental rates of nuclear transplant embryos to blastocysts were significantly(P< 0.05) higher(57.1%) than those using preactivated ones(20.8%). The cell counts of nuclear transplant embryos developed to blastosyst stage were increased signficantly(P<0.05) more in the non-preactivated recipient cytoplasm(163.7 cells), as compared whit the preactivated recipient cytoplasm(85.4 cells), A total of 49 nuclear transplant embryos were tranferrid into 5 recipient does, of which two offsprings were produced from a foster mother 31 days after embryo transfer. these results showed that the blastomeres of G1 phase and non-preactivated oocytes might be utillzed efficiently as donor nuclei and recipient cytoplasms in the nuclear transplant procedure, thought the offspring production remained still low.

• 한국수정란이식학회지
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• 제15권2호
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• pp.157-165
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• 2000

The efficiency of transgenic livestock production could be improved by early screening of transgene-integration and sexing of embryos at preimplantational stages before trasferring them into recipients. We examined the effciency of multiplex PCR analysis for the simultaneous confirmation of the trasgene and sex during the preimplantational development of bovine embryos and the possibility of green fluorescent protein(GFP) gene as a non-invasive marker for the early screening of transgenic embryos. The GFP gene was microinjected into the male pronuclei of bovine zygotes produced in vitro. The injected zygotes were co-cultured in TCM-199 containing 10% FCS with boving oviductal epithelial cells in a 5% CO2 incubator. Seventeen(13.0%) out of 136 gene-injected bovine zygotes developed by multiplex PCR analysis and the expression of GFP was detected by observing green fluorescence in embryos under a fluorescent microscope. Eight(67%) of 12 embryos at 2-cell to blastocyst stage were positive in the PCR analysis, but only two(11.8%) of 17 blastocysts expressed the GFP gene. Their sex was determined as 7 female and 5 male embryos by the PCR analysis. The results indicate that the screening of GFP gene and sex in bovine embryos by PCR analysis and fluorescence detection could be a promisible method for the preselection of transgenic embryos.